During tumor progression, cancer cells undergo dynamic transformations, including tumor growth, angiogenesis, and metastatic dissemination. There is a crucial need in cancer therapeutics to develop novel approaches that target critical, ideally multiple steps, during tumor progression. In this proposal, we have designed artificial transcription factors (ATFs) made of zinc finger (ZF) domains as novel therapeutic strategy to inhibit multiple processes during tumor progression. We have targeted the mammary serine protease inhibitor (maspin) gene (SERPINB5), Maspin is an important therapeutic target since its overexpression is associated with tumor suppression, decreased angiogenesis, motility and metastasis in breast and prostate tumor models. Metastatic cells have developed several mechanisms to down-regulate maspin. Maspin is rarely mutated in aggressive tumors. Instead, silencing of maspin involves both, transcriptional regulation and aberrant promoter methylation. Our objective is to construct ATFs able to specifically re-activate maspin in metastatic breast cell lines, overcoming epigenetic silencing. Our hypothesis is that ATFs up-regulating maspin, by themselves or in combination with drugs that increase chromatin accessibility (methyltransferase and histone deacetylase inhibitors), will be able to re-activate maspin functions in tumor cells, reduce tumor growth and metastatic spread in nude mice. First, we have engineered highly specific ATFs made of 6ZF domains targeting 18-base pairs (bp) sites in the maspin promoter. The ZFs are linked to a potent transcriptional activator domain and expressed in tumor cells using retroviral vectors. We will investigate if the ATFs are able to specifically reactivate maspin in several breast cancer cells comprising a methylated and silenced maspin promoter. We will study if the ATFs synergize with methyltransferase and histone deacetylase inhibitors, to up-regulate maspin. We will assess if these ATFs are able to down-regulate cell invasion and to induce apoptosis in metastatic cell lines. Finally, the ATFs will be expressed using Adeno- Associated Viruses (AAVs) and we will study their capability to down-regulate tumor growth and metastasis in an orthotopic xenograft model of breast cancer in nude mice. Metastatic lesions will be monitored in real time using Bioluminescence Imaging (BLI). This work should lead to the characterization of novel anti-cancer regulators, able to reprogram the aberrant epigenetic silencing of tumor suppressors [unreadable] [unreadable] [unreadable] [unreadable]